Rat liver glutathione S-transferases. DNA sequence analysis of a Yb2 cDNA clone and regulation of the Yb1 and Yb2 mRNAs by phenobarbital.

We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione S-transferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Yb1 cDNA clone, pGTA/C44, described previously by our laboratory (Ding, G. J.-F., Lu, A. Y. H., and Pickett, C. B. (1985) J. Biol. Chem. 260, 13268-13271). Over the protein coding region of the Yb1 and Yb2 cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb2 subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Yb1 Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Yb1 subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Yb1, Yb2, and Yc mRNAs. Our results clearly show that the Yb1 and Yb2 mRNAs are elevated approximately 5-6-fold by phenobarbital administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic. Finally, our data suggest that the Yb2 subunit is encoded by a gene(s) which is distinct from the Yb1 gene(s) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat.

We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione Stransferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Ybl cDNA clone, pGTA/C44, described previously by our laboratory (Ding,  J. Biol. Chern. 260, 13268-13271). Over the protein coding region of the Ybl and Y b z cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb, subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Ybl Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Ybl subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Ybl, Ybz, and Yc mRNAs. Our results clearly show that the Ybl and Ybz mRNAs are elevated -5-6fold by phenobarbitasl administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic.
Finally, our data suggest that the Y b , subunit is encoded by a gene(s) which is distinct from the Ybl genets) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat.
The rat liver glutathione S-transferases are a family of enzymes which catalyze the conjugation of the reduced sulfhydryl group of glutathione with electrophiles. In addition the transferases bind with high affinity various exogenous hydrophobic compounds as well as potentially toxic compounds such as bilirubin and heme (1-3). The enzymes are comprised of binary combinations of at least six major subunits, Ya, Ya, Ybl, Ybz, Yc, and Yn which can be separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-6).
Our laboratory has reported recently the construction and characterization of Ya, Yc, and Ybl cDNA clones (7)(8)(9). We * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
11 To whom correspondence should be addressed. have used these clones in RNA blot hybridization and nuclear run-off assays to demonstrate that the rat liver glutathione S-transferase genes are transcriptionally activated by phenobarbital and 3-methylcholanthrene (7,10). DNA sequence analysis has indicated that the Ya and Yc mRNAs have a 75% sequence homology in the protein coding region; however, the 5' and 3' untranslated regions are divergent (8). Based on the extent of sequence homology between the Ya and Yc mRNAs, we have concluded that the Ya and Yc genes are members of the same glutathione S-transferase gene family.
In contrast, however, we have found that the nucleotide sequence of the Ybl cDNA clone, pGTA/C44, is not homologous to the nucleotide sequence of the Ya or Yc cDNA clones (9). These data indicate that the Ybl subunit is encoded by a glutathione S-transferase gene family which is distinct from the Ya-Yc gene family. In the present study, we have extended our work on the Yb gene family and have isolated and characterized a new Yb cDNA clone, pGTA/C48, which is complementary to the glutathione S-transferase Y b z mRNA. Conventional protein sequence analysis of glutathione S-transferase C, a Ybl Ybz heterodimer, has confirmed the identity of pGTA/C48 as a Yb, cDNA clone. Since nucleotide sequence analysis has allowed us to ascertain regions between the glutathione S-transferase cDNA clones which lack sequence homology, we have used the divergent regions to examine the degree of induction of specific glutathione S-transferase mRNAs by phenobarbital. Both the Ybl and Y b z mRNAs are elevated in response to xenobiotie administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic.

Preparation of cDNA and Construction of Recombinant Phmids-
In order to prepare double-stranded cDNA, the glutathione S-transferase yb mRNAs were purified by polysomal immunoabsorption techniques as described previously (8). Both the first and second strands of the cDNA were synthesized as described by Gubler and Hoffman (11) and tailed with dCTP using terminal deoxynucleotidyltransferase as described previously (8).
Hybrid-select Translation-Hybrid-select translations were carried out according to the procedure described by Cleveland et al. (12) as outlined in a previous publication from our laboratory (7).
In Vitro Labeling of cDNAs-cDNAs were labeled in vitro either with [32P]dCTP by nick translation, at the 5' end with L3'P]ATP by T4 polynucleotide kinase, or at the 3' end with [32P]didexoy-ATP by terminal deoxynucleotidyltransferase (13).
Restriction Endonuclease Mapping of pGTAIC48-A restriction map of the cDNA insert in pGTA/C48 was constructed by the method of Smith and Birnstiel (14) using 5' end-labeled fragments. The sizes of the end-labeled fragments generated by the partial restriction endonuclease digestion were determined on 5% polyacrylamide gels.
Nucleotide Sequence Amlysis-The chemical method of Maxam and Gilbert was used for DNA sequence analysis (15). Appropriate endonuclease map of pGTA/C48 was determined by single and double digests as well as by partial digestion of end-labeled fragments using the Smith and Birnstiel procedure (14). The dashed lines in the clone represent pBR322 sequences. The 5' to 3' orientation of the cDNA insert in pGTA/C48 is from the BulI site towards the NarI site. The arrows represent the direction from each restriction endonuclease site that DNA sequencing proceeded. Restriction endonuclease sites used for 5' end labeling were NcoI, HindIII, NurI, and EcoRV. Restriction endonuclease sites used for 3' end labeling were the PstI sites. The sequence of all fragments were determined three to four times and approximately 70% of the sequence has been determined in both directions. restriction fragments were 5' and 3' end-labeled and subjected to DNA sequence analysis. RNA Slot Blot Analysis-Total rat liver RNA was isolated from the livers of 3-4 rats (male Sprague-Dawley) by the procedure of Chirgwin et al. (16). Poly(A+) RNA was isolated from total RNA as described previously (17). Various concentrations of poly(A+) RNA were spotted onto nitrocellulose sheets using a minifold 11-slot blot apparatus (Schleicher & Schuell). Prehybridization and hybridization of the radiolabeled cDNA probes were done as described by Thomas (18). Autoradiographs of the x-ray films were scanned with a densitometer (Bio-Rad).

MW
Amino Acid Sequence Analysis-Five nmol of the rat liver glutathione S-transferase Y b l Y b , heterodimer were sequenced in the Applied Biosystems gas phase sequenator (model 870-A) according to the manufacturer's specifications. High performance liquid chromatography was used to quantitate the phenylthiohydantoin derivatives produced at each step (19).

RESULTS
Construction and Characterization of a cDNA Clone Complementary to Rat Liver Glutathione S-Transferase Yb, mRNA-In previous work from our laboratory, we reported the isolation and characterization of a nearly full-length cDNA clone, pGTA/C44, which is complementary to the Yb, mRNA. During the screening of the Yb cDNA library, a second clone, pGTA/C48, was identified that had a restriction endonuclease map distinct from pGTA/C44 (Fig. 1). However, in hybrid-select translation experiments, pGTA/C48 hybridized to a mRNA that upon translation yielded a polypeptide with the same electrophoretic mobility as the Yb, polypeptide (Fig. 2). Since this clone cross-hybridizes with pGTA/C44, we felt it would be a likely candidate for a Y b , cDNA clone.
DNA Sequence Analysis of pGTA/C48"Nucleotide se- The entire nucleotide sequence of pGTA/C48 along with the nucleotide sequence of pGTA/C44 is presented in Fig. 3 unit is 25,705. The carboxyl-terminal sequence, Pro-Lys, agrees with one possible carboxyl terminal sequence deduced from purified glutathione S-transferase Yb dimers (20). NHz-terminal Sequence Analysis of the Rat Liver Glutathione S-Transferase Ybl Yb, Heterodimer-Since the Ybz cDNA clone was truncated and did not contain sequences complementary to the first 24 amino acids of the Y b z subunit that had been published by Prey et al.   Regulation of the Ybl, Ybz, and Yc mRNAs by Phenobarbital-Nucleotide sequence analysis of the glutathione S-transferase Ybl, Yb,, and Yc cDNA clones has allowed us to define regions that lack homology between the various cDNA inserts. The divergent regions were isolated and used as specific probes for each mRNA. To generate a specific probe from the Yb, clone, pGTA/C48 was digested with NurI. NurI cleaves at nucleotide 658 in the 3' untranslated region of the Ybz cDNA insert and three times in pBR322, generating four fragments. The largest fragment, -2600 bp, contains 187 bp of the 3' untranslated region and is a specific probe for the Y b , mRNA. To generate a specific probe from the Yb, clone, pGTA/C44 was digested with BurnHI. BarnHI cleaves at nucleotide 739 in the 3' untranslated region of the Ybl cDNA insert and once in pBR322, generating two fragments. The smallest fragment (-1450 bp) contains 299 bp of 3' untranslated region and is a specific probe for Y b , mRNA. The Yb, and Yb, probes generated by these digestions do not crosshybridize on Southern blots (data not shown). Finally, to generate a specific probe from the Yc clone, pGTB42 was digested with HindIII. HindIII cleaves at nucleotide 757 in the 3' untranslated region of the Yc cDNA insert and once in pBR322, generating two fragments. The largest fragment, -3700 bp contains 108 bp of the 3' untranslated region of the Yc mRNA. This region is specific for the Yc mRNA and does not cross-hybridize to the Ya cDNA clone, pGTB38, on Southern blots (data not shown).
The Ybl, Ybz, and Yc probes were utilized in RNA slot blots to determine the level of the glutathione S-transferase mRNAs after phenobarbital administration. The Ybl and Ybz mRNAs were elevated in response to phenobarbital administration, reaching a maximum induction, -&"-fold, at 16-24 h (Table 11). Interestingly, the Yc mRNA level was only elevated -2-fold at 16 h (Table 11).

DISCUSSION
We have constructed and characterized a cDNA clone, pGTA/C48, which is complementary to the Yb, mRNA. In the protein coding region the nucleotide sequence of pGTA/ C48 is 84% homologous to the nucleotide sequence of the Yb, cDNA clone, pGTA/C44, described previously by our laboratory (9). However, the 3' untranslated regions of the Ybz and the Yb, clones are only 32% homologous. These data suggest that the Ybl and Ybz polypeptides are encoded by two different genes rather than being generated by alternative processing of a single gene.
Nucleotide sequence analysis of the Ybl and Y b z cDNA clones have enabled us to identify regions of poor homology in the 3' untranslated regions of the two mRNAs. We have used these regions as specific probes in RNA slot blot analysis to demonstrate that both the Ybl and Y b 2 mRNAs are elevated by phenobarbital administration. These data are in contrast with previous reports indicating that only the glutathione S-transferase Ybl subunit is elevated by phenobarbital administration (4,20,22,23). The reason for this discrepancy is unclear. However, ideally, the effect of phenobarbital on the Y b z subunit should be examined using immunochemical procedures. Unfortunately, the existent polyclonal antibodies are not specific enough to distinguish different Yb subunits. It is unclear whether the changes in the Y b z mRNA correlate with an increase in Y b z protein. Although the Ya mRNA is elevated -8-fold by phenobarbital administration (7), the Yc mRNA is reported to be unaffected by phenobarbital treatment or only slightly elevated (17, 24, 25). However these data were based exclusively on in uitro translation analysis and may have been due to differences in translational efficiencies of the Yc mRNA versus the Ya mRNA. Interestingly, our hybridization assay with a specific Yc probe showed that phenobarbital resulted in a very modest elevation in the Yc mRNA, which is consistent with our previous in vitro translation and immunoprecipitation experiments (24). Therefore, only the Ya, Ybl and Y b z mRNAs are elevated significantly by phenobarbital. This elevation is due in part to transcriptional activation of the Ya and Yb genes (10).
The rat liver Ybl cDNA clone, pGTA/C44, shares significant nucleotide sequence homology with a mouse liver glutathione S-transferase cDNA clone, pGT55, characterized by Pearson et al. (26). Although pGTB55 is complementary to a mouse liver glutathione S-transferase mRNA encoding a polypeptide with an isoelectric point of 9.3, it shares significant sequence homology with a second mouse liver glutathione Stransferase mRNA encoding a polypeptide with an isoelectric point of 8.7. The rat liver Y b , subunit may represent the analog of the mouse liver glutathione S-transferase 8.7 subunit. The NHz-terminal sequence of a human glutathione Stransferase (27) is homologous to the NH2-terminal sequence of the rat liver Y b l and Ybz subunits. Although it is unknown whether the sequence homology between the human liver glutathione S-transferase and the rat liver glutathione Stransferases will be conserved throughout the entire amino acid sequence, the similarity in the NH,-terminal sequences suggests that these glutathione S-transferases have evolved from a common ancestral gene.
The existence of two Yb mRNAs in rat liver may represent the minimal number present. Tu and Reddy (28) have indicated that five different Yb homodimers or heterodimers can be isolated from rat liver cytosol. Interestingly, Hayes (6) has characterized a glutathione S-transferase subunit, designated Yn, which can form a heterodimer with the Ybl or Y b z subunit. The Yn subunit shares a number of common tryptic peptides with the Ybl and Y b z subunits, suggesting they have regions of amino acid sequence homology. We have not identified a third cDNA clone in our cDNA library that is complementary to the Yn mRNA; however, this is not surprising given the very low level of the Yn subunit and presumably the mRNA in rat liver (6).
The isolation and characterization of a cDNA clone complementary to the Y h , mRNA provide further evidence for heterogeneity in the rat liver glutathione S-transferase multigene family. The YbZ cDNA clone will allow us to isolate the structural gene(s) encoding the glutathione S-transferase Y b z subunit as well as elucidate the mechanism(s) by which this gene is regulated by xenobiotics.